Feasibility and Safety of Personalized, Multi-Target, Adoptive Cell Therapy (IMA101): First-in-Human Clinical Trial in Patients with Advanced Metastatic Cancer.

IMA101 is an actively personalized, multi-targeted adoptive cell therapy (ACT), whereby autologous T cells are directed against multiple novel defined peptide-HLA (pHLA) cancer targets. HLA-A*02:01-positive patients with relapsed/refractory solid tumors expressing ≥1 of 8 predefined targets underwent leukapheresis. Endogenous T cells specific for up to 4 targets were primed and expanded in vitro. Patients received lymphodepletion (fludarabine, cyclophosphamide), followed by T-cell infusion and low-dose IL2 (Cohort 1). Patients in Cohort 2 received atezolizumab for up to 1 year (NCT02876510). Overall, 214 patients were screened, 15 received lymphodepletion (13 women, 2 men; median age, 44 years), and 14 were treated with T-cell products. IMA101 treatment was feasible and well tolerated. The most common adverse events were cytokine release syndrome (Grade 1, n = 6; Grade 2, n = 4) and expected cytopenias. No patient died during the first 100 days after T-cell therapy. No neurotoxicity was observed. No objective responses were noted. Prolonged disease stabilization was noted in three patients lasting for 13.7, 12.9, and 7.3 months. High frequencies of target-specific T cells (up to 78.7% of CD8+ cells) were detected in the blood of treated patients, persisted for >1 year, and were detectable in posttreatment tumor tissue. Individual T-cell receptors (TCR) contained in T-cell products exhibited broad variation in TCR avidity, with the majority being low avidity. High-avidity TCRs were identified in some patients' products. This study demonstrates the feasibility and tolerability of an actively personalized ACT directed to multiple defined pHLA cancer targets. Results warrant further evaluation of multi-target ACT approaches using potent high-avidity TCRs. See related Spotlight by Uslu and June, p. 865.

An ultra-high-throughput screen for the evaluation of peptide HLA-Binder interactions.

Peptide human leukocyte antigen (pHLA) targeting therapeutics like T-cell receptor based adoptive cell therapy or bispecific T cell engaging receptor molecules hold great promise for the treatment of cancer. Comprehensive pre-clinical screening of therapeutic candidates is important to ensure patient safety but is challenging because of the size of the potential off-target space. By combining stabilized peptide-receptive HLA molecules with microarray printing and screening, we have developed an ultra-high-throughput screening platform named ValidaTe that enables large scale evaluation of pHLA-binder interactions. We demonstrate its potential by measuring and analyzing over 30.000 binding curves for a high-affinity T cell Engaging Receptor towards a large pHLA library. Compared to a dataset obtained by conventional bio-layer interferometry measurements, we illustrate that a massively increased throughput (over 650 fold) is obtained by our microarray screening, paving the way for use in pre-clinical safety screening of pHLA-targeting drugs.

Quantitative immunopeptidomics reveals a tumor stroma-specific target for T cell therapy.

T cell receptor (TCR)-based immunotherapy has emerged as a promising therapeutic approach for the treatment of patients with solid cancers. Identifying peptide-human leukocyte antigen (pHLA) complexes highly presented on tumors and rarely expressed on healthy tissue in combination with high-affinity TCRs that when introduced into T cells can redirect T cells to eliminate tumor but not healthy tissue is a key requirement for safe and efficacious TCR-based therapies. To discover promising shared tumor antigens that could be targeted via TCR-based adoptive T cell therapy, we employed population-scale immunopeptidomics using quantitative mass spectrometry across ~1500 tumor and normal tissue samples. We identified an HLA-A*02:01-restricted pan-cancer epitope within the collagen type VI α-3 (COL6A3) gene that is highly presented on tumor stroma across multiple solid cancers due to a tumor-specific alternative splicing event that rarely occurs outside the tumor microenvironment. T cells expressing natural COL6A3-specific TCRs demonstrated only modest activity against cells presenting high copy numbers of COL6A3 pHLAs. One of these TCRs was affinity-enhanced, enabling transduced T cells to specifically eliminate tumors in vivo that expressed similar copy numbers of pHLAs as primary tumor specimens. The enhanced TCR variants exhibited a favorable safety profile with no detectable off-target reactivity, paving the way to initiate clinical trials using COL6A3-specific TCRs to target an array of solid tumors.

SARS-CoV-2-reactive T-cell receptors isolated from convalescent COVID-19 patients confer potent T-cell effector function.

Both B cells and T cells are involved in an effective immune response to SARS-CoV-2, the disease-causing virus of COVID-19. While B cells-with the indispensable help of CD4+ T cells-are essential to generate neutralizing antibodies, T cells on their own have been recognized as another major player in effective anti-SARS-CoV-2 immunity. In this report, we provide insights into the characteristics of individual HLA-A*02:01- and HLA-A*24:02-restricted SARS-CoV-2-reactive TCRs, isolated from convalescent COVID-19 patients. We observed that SARS-CoV-2-reactive T-cell populations were clearly detectable in convalescent samples and that TCRs isolated from these T cell clones were highly functional upon ectopic re-expression. The SARS-CoV-2-reactive TCRs described in this report mediated potent TCR signaling in reporter assays with low nanomolar EC50 values. We further demonstrate that these SARS-CoV-2-reactive TCRs conferred powerful T-cell effector function to primary CD8+ T cells as evident by a robust anti-SARS-CoV-2 IFN-γ response and in vitro cytotoxicity. We also provide an example of a long-lasting anti-SARS-CoV-2 memory response by reisolation of one of the retrieved TCRs 5 months after initial sampling. Taken together, these findings contribute to a better understanding of anti-SARS-CoV-2 T-cell immunity and may contribute to paving the way toward immunotherapeutics approaches targeting SARS-CoV-2.

Optimization in Detection of Antigen-Specific T Cells Through Differentially Labeled MHC Multimers.

A large variety of fluorescent molecules are used on a regular basis to tag major histocompatibility complex (MHC) multimers for detection of antigen-specific T cells. We have evaluated the way in which the choice of fluorescent label can impact the detection of MHC multimer binding T cells in an exploratory proficiency panel where detection of MHC multimer binding T cells was assessed across 16 different laboratories. We found that the staining index (SI) of the multimer reagent provided the best direct correlation with the value of a given fluorochrome for T cell detection studies. The SI is dependent on flow cytometer settings and chosen antibody panel; hence, the optimal fluorochrome selection may differ from lab to lab. Consequently, we describe a strategy to evaluate performance of the detection channels and optimize the SI for selected fluorescent molecules. This approach can easily be used to test and optimize fluorescence detection in relation to MHC multimer staining and in general, for antibody-based identification of rare cell populations. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

High-throughput peptide-MHC complex generation and kinetic screenings of TCRs with peptide-receptive HLA-A*02:01 molecules.

Major histocompatibility complex (MHC) class I molecules present short peptide ligands on the cell surface for interrogation by cytotoxic CD8+ T cells. MHC class I complexes presenting tumor-associated peptides such as neoantigens represent key targets of cancer immunotherapy approaches currently in development, making them important for efficacy and safety screenings. Without peptide ligand, MHC class I complexes are unstable and decay quickly, making the production of soluble monomers for analytical purposes labor intensive. We have developed a disulfide-stabilized HLA-A*02:01 molecule that is stable without peptide but can form peptide-MHC complexes (pMHCs) with ligands of choice in a one-step loading procedure. We illustrate the similarity between the engineered mutant and the wild-type molecule with respect to affinity of wild-type or affinity-matured T cell receptors (TCRs) and present a crystal structure corroborating the binding kinetics measurements. In addition, we demonstrate a high-throughput binding kinetics measurement platform to analyze the binding characteristics of bispecific TCR (bsTCR) molecules against diverse pMHC libraries produced with the disulfide-stabilized HLA-A*02:01 molecule. We show that bsTCR affinities for pMHCs are indicative of in vitro function and generate a bsTCR binding motif to identify potential off-target interactions in the human proteome. These findings showcase the potential of the platform and the engineered HLA-A*02:01 molecule in the emerging field of pMHC-targeting biologics.

Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.

Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users to generate large-scale TERS, on-site using the most commonly used electroporation (EP) devices, an RNA-based kit approach, providing stable TCR mRNA and an optimized manufacturing protocol were established. In preparation for the release of this immuno-control kit, we established optimal EP conditions on six devices and initiated an extended RNA stability study. Furthermore, we coordinated on-site production of TERS with 4 participants. Finally, a proficiency panel was organized to test the unsupervised production of TERS at different laboratories using the kit approach. The results obtained show the feasibility and robustness of the kit approach for versatile in-house production of cellular control samples.

Long-term follow-up after left atrial appendage occlusion with comparison of transesophageal echocardiography versus computed tomography to guide medical therapy and data about postclosure cardioversion.

AIMS: The use of left atrial appendage (LAA) occluders in atrial fibrillation is increasing. There are few data on the comparison between transesophageal echocardiography (TEE) and computed tomography (MDCT) assessing peridevice flow and outcome of electrical cardioversion (ECV) in these patients. METHODS AND RESULTS: Single-center prospective registry from 2009 to 2015 including all LAA occluders to analyze success and complications during implantation and follow-up. Patients having ≥1 ECV were further analyzed. TEE was performed during implantation and at 6 weeks. In a subgroup of 77 patients, we compared MDCT with TEE at 6 weeks. Overall, 135 patients (69 ± 9 years; 70% male; CHA2 DS2 -VASc score: 3.6 ± 1.4; HAS-BLED score: 2.5 ± 0.6) received a LAA occluder (Watchman, n = 73; ACP-1, n = 59; Amulet, n = 3; PVI + LAA occluder, n = 91; and LAA occluder only, n = 44). Device implantation was successful in 131 (97%). Eight patients (5.9%) had major periprocedural complications (ischemic stroke/transient ischemic attacks, n = 4, tamponade, n = 2, device thrombosis, n = 2, Dressler syndrome, n = 1). The periprocedural complication rate was similar between concomitant procedure and LAA occluder only (8/91 vs. 5/44; P = 0.6). Twelve patients (9%) died (procedure-related, n = 2; 1%) during follow-up of 44 months (IQR: 43). MDCT (n = 77) at 6 weeks showed similar peridevice flow compared to TEE (TEE: 1.5 ± 1.9 mm vs. MDCT: 1.1 ± 2.2 mm, P = 0.25). Thromboembolic events occurred in 3 patients (CVA, n = 1; TIA, n = 2) during follow-up. In total, 41 ECV were performed in 26 patients (1.6 ± 0.9/patient), 13 months (IQR: 24) after implantation (<1 month: n = 8). No ECV-related clinical complications were observed. CONCLUSION: LAA occlusion is feasible with an acceptable safety profile and few events during long-term follow-up. ECV after LAA occlusion is feasible. MDCT could help to evaluate peridevice flow.

IMA901, a multipeptide cancer vaccine, plus sunitinib versus sunitinib alone, as first-line therapy for advanced or metastatic renal cell carcinoma (IMPRINT): a multicentre, open-label, randomised, controlled, phase 3 trial.

BACKGROUND: In a phase 2 study in patients with metastatic renal cell carcinoma, overall survival was associated with T-cell responses against IMA901, a vaccine consisting of ten tumour-associated peptides. In this phase 3 trial, we aimed to determine the clinical effect of adding IMA901 to sunitinib, the standard first-line treatment in metastatic renal cell carcinoma with postulated favourable immunomodulatory effects. METHODS: The IMPRINT study is an open-label, randomised, controlled, phase 3 trial done at 124 clinical sites in 11 countries. HLA-A*02-positive patients (aged ≥18 years) with treatment-naive, histologically confirmed metastatic or locally advanced (or both) clear-cell renal cell carcinoma were randomly assigned (3:2) to receive sunitinib plus up to ten intradermal vaccinations of IMA901 (4·13 mg) and granulocyte macrophage colony-stimulating factor (75 µg), with one dose of cyclophosphamide (300 mg/m2) 3 days before the first vaccination, or to receive sunitinib alone. Sunitinib (50 mg) was given orally once daily, with each cycle defined as 4 weeks on treatment followed by 2 weeks off treatment, until progression of disease as determined by the investigator, death, or withdrawal of consent. Block randomisation (block size five) was done centrally using an interactive web response system, stratified by prognostic risk, geographical region, and previous nephrectomy. Patients and investigators were not masked to treatment allocation. The primary endpoint was overall survival from randomisation until death of any cause as determined by the investigator, analysed by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01265901. FINDINGS: Between Dec 22, 2010, and Dec 15, 2012, we screened 1171 patients, of whom 339 were randomly assigned to receive sunitinib plus IMA901 (n=204) or sunitinib monotherapy (n=135). Patients had a median follow-up of 33·27 months (IQR 29·92-35·64). Median overall survival did not differ significantly between the groups (33·17 months [95% CI 27·81-41·36] in the sunitinib plus IMA901 group vs not reached [33·67-not reached] in the sunitinib monotherapy group; hazard ratio 1·34 [0·96-1·86]; p=0·087). 116 (57%) of 202 patients in the sunitinib plus IMA901 group and 62 (47%) of 132 in the sunitinib group had grade 3 or worse adverse events, the most common of which were hypertension, neutropenia, and anaemia in both groups, and mild-to-moderate transient injection-site reactions (eg, erythema, pruritus) were the most frequent IMA901-related side-effect in the sunitinib plus IMA901 group. Serious adverse events leading to death occurred in four (2%) patients (one respiratory failure and circulatory collapse [possibly related to sunitinib], one oesophageal varices haemorrhage [possibly related to sunitinib], one cardiac arrest [possibly related to sunitinib], and one myocardial infarction) and eight (6%) patients in the sunitinib group (one case each of renal failure, oesophageal varices haemorrhage, circulatory collapse, wound infection, ileus, cerebrovascular accident [possibly treatment related], and sepsis). INTERPRETATION: IMA901 did not improve overall survival when added to sunitinib as first-line treatment in patients with metastatic renal cell carcinoma. The magnitude of immune responses needs to be improved before further development of IMA901 in this disease is indicated. FUNDING: Immatics Biotechnologies.

Toward harmonized phenotyping of human myeloid-derived suppressor cells by flow cytometry: results from an interim study.

There is an increasing interest for monitoring circulating myeloid-derived suppressor cells (MDSCs) in cancer patients, but there are also divergences in their phenotypic definition. To overcome this obstacle, the Cancer Immunoguiding Program under the umbrella of the Association of Cancer Immunotherapy is coordinating a proficiency panel program that aims at harmonizing MDSC phenotyping. After a consultation period, a two-stage approach was designed to harmonize MDSC phenotype. In the first step, an international consortium of 23 laboratories immunophenotyped 10 putative MDSC subsets on pretested, peripheral blood mononuclear cells of healthy donors to assess the level of concordance and define robust marker combinations for the identification of circulating MDSCs. At this stage, no mandatory requirements to standardize reagents or protocols were introduced. Data analysis revealed a small intra-laboratory, but very high inter-laboratory variance for all MDSC subsets, especially for the granulocytic subsets. In particular, the use of a dead-cell marker altered significantly the reported percentage of granulocytic MDSCs, confirming that these cells are especially sensitive to cryopreservation and/or thawing. Importantly, the gating strategy was heterogeneous and associated with high inter-center variance. Overall, our results document the high variability in MDSC phenotyping in the multicenter setting if no harmonization/standardization measures are applied. Although the observed variability depended on a number of identified parameters, the main parameter associated with variation was the gating strategy. Based on these findings, we propose further efforts to harmonize marker combinations and gating parameters to identify strategies for a robust enumeration of MDSC subsets.

Cryopreservation of MHC multimers: Recommendations for quality assurance in detection of antigen specific T cells.

Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5-16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability.

Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

Clinical Long-Term Response to Cardiac Resynchronization Therapy Is Independent of Persisting Echocardiographic Markers of Dyssynchrony.

BACKGROUND: The aim of the study was to prove the concept that correction of established parameters of dyssynchrony is a requirement for favorable long-term outcome in patients with cardiac resynchronization therapy (CRT), whereas patients with persisting dyssynchrony should have a less favorable response. METHODS: After CRT implantation and optimization of dyssynchrony parameters, we evaluated whether correction or persistence of dyssynchrony predicted long-term outcome. Primary endpoint was a combination of cardiac mortality/heart transplantation and hospitalization due to worsening heart failure, and secondary endpoint was NYHA class. RESULTS: One hundred twenty-eight consecutive patients (mean age 68 ± 10 years) undergoing CRT with a mean left ventricular ejection fraction of 27±9% were followed for 27 ± 19 months. All cause mortality was 17.2%, cardiac mortality was 7.8% and 3.1% had to undergo heart transplantation. Rehospitalization due to worsening heart failure was observed in 14.8%. NYHA class before CRT implantation was 2.8 ± 0.8 and improved during follow-up to 2.0 ± 0.8 (P < 0.001). A clinical response was observed in 76% (n = 97) and an echocardiographic response was documented in 66% (n = 85). After individually optimized AV and VV intervals with echocardiography, atrioventricular dyssynchrony was still present in 7.2%, interventricular dyssynchrony in 13.3% and intraventricular dyssynchrony in 16.4%. Despite persistent atrioventricular, interventricular and intraventricular dyssynchrony at long-term follow-up, the combined primary and secondary endpoints did not differ compared to the group without mechanical dyssynchrony (P = ns). QRS duration with biventricular stimulation did not differ between responders vs. nonresponders. CONCLUSION: After successful CRT implantation, clinical long-term response is independent of correction of dyssynchrony measured by echocardiographic parameters and QRS width.

Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival.

IMA901 is the first therapeutic vaccine for renal cell cancer (RCC) consisting of multiple tumor-associated peptides (TUMAPs) confirmed to be naturally presented in human cancer tissue. We treated a total of 96 human leukocyte antigen A (HLA-A)*02(+) subjects with advanced RCC with IMA901 in two consecutive studies. In the phase 1 study, the T cell responses of the patients to multiple TUMAPs were associated with better disease control and lower numbers of prevaccine forkhead box P3 (FOXP3)(+) regulatory T (T(reg)) cells. The randomized phase 2 trial showed that a single dose of cyclophosphamide reduced the number of T(reg) cells and confirmed that immune responses to multiple TUMAPs were associated with longer overall survival. Furthermore, among six predefined populations of myeloid-derived suppressor cells, two were prognostic for overall survival, and among over 300 serum biomarkers, we identified apolipoprotein A-I (APOA1) and chemokine (C-C motif) ligand 17 (CCL17) as being predictive for both immune response to IMA901 and overall survival. A randomized phase 3 study to determine the clinical benefit of treatment with IMA901 is ongoing.

Intravenous enoxaparin versus unfractionated heparin in unselected patients undergoing percutaneous coronary interventions: the Zurich enoxaparin versus unfractionated heparin in PCI study (ZEUS).

AIMS: To evaluate the efficacy and safety of intravenous enoxaparin as an alternative to unfractionated heparin (UFH) as antithrombotic therapy in unselected patients undergoing percutaneous coronary intervention (PCI). METHODS AND RESULTS: Eight hundred and seventy-six (876) consecutive eligible patients undergoing PCI were prospectively randomised to either intravenous enoxaparin 0.75 mg/kg or dose-adjusted UFH in this open-label study that was prematurely stopped due to slow recruitment. Randomisation was stratified on elective PCI or PCI for acute coronary syndrome (ACS). The primary endpoint was a combination of death, myocardial infarction, unplanned target vessel revascularisation and major bleeding at 30 days. Secondary endpoint was a composite of major and minor bleeding and thrombocytopenia < 50x109. The primary endpoint of intravenous enoxaparin did not differ from those of UFH (5.5% vs. 7.0%, p=ns) whereas safety endpoints were reduced with enoxaparin compared to UFH (9.9% vs. 20.0%, p<0.001). Among 229 (26%) patients presenting with ACS, the incidence of both, the primary and secondary endpoints, was lower with enoxaparin as compared to UFH (1.8% vs. 12.9% and 14.2% vs. 31%, p<0.001 and p=0.003, respectively). CONCLUSIONS: Due to the premature halting of the study and the low event rate, these data are observational only, and no definite conclusion could be made concerning efficacy and safety of intravenous enoxaparin as an alternative to UFH in unselected patients undergoing PCI.

Interaction of vitamin K antagonists with heparin affect monitoring by activated clotting times.

BACKGROUND: Unfractionated heparin is recommended during atrial fibrillation (AF) ablation to achieve activated clotting time (ACT) above 250-300 s to prevent clot. Many patients on therapeutic international normalised ratio (INR) undergo AF ablation procedures; however, it is unknown whether they require less heparin to achieve similar ACT levels. METHODS: During AF ablation, the ACT was measured before and 10 min after administration of i.v. unfractionated heparin in patients with and without anticoagulation. The association of INR, heparin, pre-procedure ACT and body weight with ACT after heparin administration was tested using multivariable linear regression models. RESULTS: The subjects of this study were 149 patients undergoing AF ablation, among them 40 (27%) with subtherapeutic INR < 2, 79 (53%) with an INR between 2 and 3, and 30 (20%) patients with INR > 3. Baseline ACT was associated with INR (r = 0.33, p < 0.001). After a mean of 8,685 +/- 2,015 U (range, 5,000-15,000 IU) unfractionated heparin, univariate predictors of ACT were baseline INR (p < 0.001), heparin dose (p = 0.012) and baseline ACT (p = 0.027). In the multivariable model, baseline INR (part r = 0.64, p < 0.001) and heparin dose (part r = 0.33, p < 0.001) strongly predicted post-heparin ACT. Estimated from the regression model, the heparin dose reductions by approximately one third in those with an INR of 2-3 and by at least two thirds in those with an INR above 3 may be favourable. Over the following 3 months, no thromboembolism and acute bleeding were observed. CONCLUSION: The INR was the strongest predictor of post-heparin ACT, even more important than the heparin dose itself. The reduction of heparin dose by one third if INR is between 2-3 and by two thirds if INR is above 3 may be favourable.

Long-term survival and functional outcome of unselected patients undergoing percutaneous coronary intervention for acute myocardial infarction.

BACKGROUND: Percutaneous coronary intervention (PCI) is the most effective reperfusion modality in patients with acute myocardial infarction (MI). Data concerning long-term survival and functional outcome are sparse. METHODS: One thousand consecutive patients treated by emergency PCI were systematically ana-lysed in a single-centre registry. Multivariate predictors of in-hospital mortality, post-discharge mortality and late functional capacity were identified. RESULTS: Follow-up was completed for 978 patients. The median clinical follow-up length was 3.2 years. In-hospital and post-discharge mortality were 7.6% and 7.3%, respectively. Annualised post-discharge mortality remained stable over time at 2% per year. Independent predictors of in-hospital death were cardiogenic shock, TIMI flow <3 after PCI, left ventricular ejection fraction <40%, age and time to patent artery >6 h. Independent predictors of post-discharge mortality were TIMI flow after PCI <3, prior MI, elevated glucose levels at admission, and increasing age. In contrast, cardiogenic shock, time to patent artery and left ventricular ejection fraction <40% were not independently associated with post-hospital death. At late follow-up, 47% of patients had normal functional capacity and 49.1% were in New York Heart Association functional class II. Predictors of impaired functional capacity at follow-up were age, gender, smoking habits and multivessel coronary disease. CONCLUSIONS: Post-discharge mortality after PCI for acute MI was 2% per year. Significant differences exist between predictors of in-hospital and post-discharge mortality. The functional capacity of surviving patients was remarkably good, even when presented in cardiogenic shock.

Identification of HLA-A*01- and HLA-A*02-restricted CD8+ T-cell epitopes shared among group B enteroviruses.

Acute enteroviral infections ranging from meningitis, pancreatitis to myocarditis are common and normally well controlled by the host immune system comprising virus-specific CD8+ cytotoxic T lymphocytes (CTL). However, in some patients enteroviruses and especially coxsackieviruses of group B are capable of inducing severe chronic forms of diseases such as chronic myocarditis. Currently, it is not known whether divergences in the CTL-related immune response may contribute to the different outcome and course of enterovirus myocarditis. A pre-requisite for the study of CTL reactions in patients with acute and chronic myocarditis is the identification of CTL epitopes. In order to define dominant enterovirus CTL epitopes, we have screened, by using gamma interferon (IFN-gamma) ELISPOT, 62 HLA-A*01- and 59 HLA-A*02-positive healthy blood donors for pre-existing CTL reactions against 12 HLA-A*01 and 20 HLA-A*02 predicted CTL epitopes derived from coxsackieviruses of group B. Positive CTL reactions were verified by FACS analysis in a combined major histocompatibility complex-tetramer IFN-gamma staining. A total of 14.8% of all donors reacted against one of the three identified epitopes MLDGHLIAFDY, YGDDVIASY or GIIYIIYKL. The HLA-A*02-restricted epitope ILMNDQEVGV was recognized by 25% of all tested blood donors. For this peptide, we could demonstrate specific granzyme B secretion, a strong cytolytic potential and endogenous processing. All four epitopes were homologous in 36-92% of group B enteroviruses, providing a strong basis for monitoring the divergence of T-cell-based immune responses in enterovirus-induced acute and chronic diseases.